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Small, soluble oligomers of amyloid-β (Aβ) are now widely believed to be the most toxic form of the peptide. Could their presence in the cerebrospinal fluid (CSF) be an early sign of impending Alzheimer’s disease? That remains to be seen, unfortunately, as measuring these nefarious peptides in biological samples is notoriously difficult. Now, as detailed in the October 20 Neurology online, scientists have made some headway in measuring similar peptides in Parkinson’s disease (PD) patients. Using an antibody-based test (ELISA), researchers led by Omar El-Agnaf, United Arab Emirates University, Al-Ain, report that, compared to controls, people with PD have more α-synuclein oligomers in their CSF. Measuring the ratio of oligomeric to total synuclein improved the sensitivity of the test. People with the mildest symptoms or who were diagnosed recently, that is, within a year, had the highest level of CSF oligomers and a higher oligomer-to-total synuclein ratio. “I believe this supports a role for α-synuclein oligomers in the very early stage of the disease,” El-Agnaf told ARF.

Joint first authors Takahiko Tokuda, Kyoto Prefectural University of Medicine, Japan, Mohamed M. Qureshi at UAE University, and colleagues used an oligomer-specific ELISA test previously developed by El-Agnaf and colleagues (see El-Agnaf et al., 2006). The test uses the same monoclonal antibody (mAb 211) to capture and detect α-synuclein. Because they only have one epitope for the antibody, captured monomers do not bind mAb 211 in the detection step, making the test specific for dimers and larger oligomers. The authors previously used colorimetric detection for the second antibody, but have now improved on that with a chemiluminescence-based method that is more sensitive. That change allowed them to measure α-synuclein oligomers in CSF without concentrating the samples, a process that could potentially compromise the assay.

With samples from a cohort of 32 PD patients and 28 controls, the researchers found, as previously, that total synuclein trended lower in the CSF of people with PD. In contrast, synuclein oligomer levels were significantly higher in PD CSF samples. Measuring the ratio of oligomer to total synuclein raised the specificity of the test from 87.5 to 90.6 percent, and the sensitivity from 75 percent to 89.3 percent.

“These findings indicate striking similarities between α-synuclein and Aβ as CSF biomarkers for Parkinson’s disease and Alzheimer’s disease, respectively,” wrote Lars Lannfelt, Uppsala University, Sweden, in an e-mail to ARF (see full comment below). Reduced CSF Aβ42 has long been on the radar as a potential diagnostic marker for incipient AD (see Hansson et al., 2006). Earlier this year, researchers in Japan reported that oligomers of Aβ might serve the same purpose (see ARF related news story on Fukumoto et al., 2010). Lannfelt believes that the Japanese groups of Fukumoto are the best attempt so far at measuring oligomeric Aβ in human CSF; Lannfelt’s group is currently pursuing a valid test for Aβ oligomers themselves.

Dominic Walsh, University College Dublin, Ireland, was more cautiously optimistic. He noted the one major problem with many of the tests for Aβ oligomers is that they use antibodies to the N-terminus of the peptide. “They can cross-react with APP, sAPPα, and C99, so you don’t really know that you are detecting Aβ,” he told ARF. “And those other species can be much more abundant, complicating matters even more.” Monomeric Aβ will also compete with oligomeric Aβ for those antibodies, such that lower levels of the monomer would increase the chances of detecting oligomers and by default give higher readings. “Competition effects complicate these types of ELISA,” said Walsh.

The best hope for a test for Aβ oligomers may lie in antibodies that seem to specifically recognize larger oligomeric species. Antibodies (WO1 and WO2) generated by Ron Wetzel’s group at the University of Tennessee Medical Center, Knoxville, recognize amyloid protofibrils (see O’Nuallain et al., 2002), as do antibodies generated by Lannfelt’s group (see Englund et al., 2007). Lannfelt is founder and member of the board of the Swedish company BioArctic Neuroscience AB, which is developing an Aβ protofibril-specific antibody as both a diagnostic tool (AβN) and therapeutic (BAN2401) for AD. According to BioArctic, the company just started a clinical trial of the latter in 80 AD patients.

El-Agnaf told ARF that he is also developing an antibody test for oligomeric forms of Aβ. He agrees that one of the challenges is finding the right antibody, one that binds to an epitope that is accessible when the peptide forms oligomers. Aggregates of Aβ and α-synuclein also tend to bind to other components of CSF. “Mild detergent helps to make more epitopes available and improve the signal,” he told ARF.

In an accompanying Neurology editorial, Clive Ballard and Emma Jones at King’s College London, U.K., note that specific forms of α-synuclein, such as oligomers or post-translationally modified protein, may distinguish people with synucleinopathies. These include PD, dementia with Lewy bodies (DLB), AD, and other neurodegenerative diseases characterized by synuclein aggregates. In a recent analysis of a Dutch cohort, CSF α-synuclein analysis failed to discriminate among PD, DLB, and AD (see Reesink et al., 2010). El-Agnaf’s study “is probably the most robust evidence so far supporting the potential utility of a specific α-synuclein measurement,” wrote Ballard and Jones. Though they cautioned that further validation is needed and “it is too preliminary to put this forward as a diagnostic test for PD.” El-Agnaf told ARF that he is testing the method on additional sample sets from Italy and Norway.—Tom Fagan

Comments on News and Primary Papers

The potential of CSF α-synuclein as a biomarker for Parkinson’s disease has been investigated by Tokuda et al. The authors confirm their own previously reported findings of decreased total CSF α-synuclein in patients as compared to controls. What is novel and intriguing with the current report is the finding of increased levels of oligomeric α-synuclein in CSF from Parkinson’s patients. Furthermore, an increased oligomer/total α-synuclein ratio offers an even better disease biomarker with sensitivity and specificity of approximately 90 percent. Importantly, the authors also state that the increased ratio is even more pronounced among cases at a milder disease stage.

These findings indicate striking similarities between α-synuclein and Aβ as CSF biomarkers for Parkinson’s disease and Alzheimer’s disease, respectively. Decreased CSF Aβ42, together with increased tau, has for a long time been the most robust biomarker for Alzheimer’s disease. Moreover, Fukumoto and colleagues recently showed that oligomeric Aβ is increased in CSF from Alzheimer’s patients (1).

Among several possible explanations, the combination of lowered total protein and increased oligomeric protein may be explained by the fact that monomeric α-synuclein and Aβ both are consumed as a result of fibril formation. Meanwhile, at least parts of the more toxic oligomeric forms may be off-pathway and thus remain in increased amounts throughout the CNS in the respective disorders.

The assays developed by Tokuda et al. and Fukumoto et al. probably detect a vast range of multimeric proteins, ranging from dimers to large oligomers or protofibrils. The levels of oligomeric α-synuclein were only given as arbitrary units, as it was difficult to generate a reliable standard curve for such protein species. Nevertheless, these findings are very interesting and promising, but need to be replicated by other research teams. In addition, future novel assays might be developed in order to discriminate which oligomeric species are most appropriate as disease biomarkers. Preferably, such assays could utilize antibodies selective for various oligomers of α-synuclein and Aβ, respectively.